Aldo-keto Reductase 1B10 Restrains Cell Migration, Invasion, and Adhesion of Gastric Cancer via Regulating Integrin Subunit Alpha 5

Background/Aims: Gastric cancer is a prevalent malignancy with unfavorable prognosis partially resulting from its high metastasis rate. Clarifying the molecular mechanism of gastric cancer occurrence and progression for improvement of therapeutic efficacy and prognosis is needed. The study tended to delineate the role and regulatory mechanism of aldo-keto reductase 1B10 (AKR1B10) in gastric cancer progression. Materials and Methods: The relationship of AKR1B10 expression with survival rate in gastric cancer was analyzed through Kaplan–Meier analysis. The mRNA levels of AKR1B10 and integrin subunit alpha 5 (ITGA5) in gastric cancer tissues and cell lines were measured by real-time quantitative polymerase chain reaction. Protein levels of AKR1B10 and integrin subunit alpha 5 were assayed via western blot. The molecular relationship between AKR1B10 and ITGA5 was analyzed by co-immunoprecipitation assay. Cell viability was assayed through Cell Counting Kit-8, invasion and migration of tumor cells was assessed through wound healing and transwell assays. Transwell assay was utilized to detect invasion. The adhesion of gastric cancer cells was detected using cell adhesion assays. Results: The results unveiled that integrin subunit alpha 5 was upregulated, while AKR1B10 was downregulated in gastric cancer tissues and cells. Overexpressing AKR1B10 hindered gastric cancer cell proliferation, migration, invasion and adhesion. It was striking that we certified the inhibitory effect of AKR1B10 on integrin subunit alpha 5 expression and their (AKR1B10 and ITGA5)) negative relationship via bioinformatics method, real-time quantitative polymerase chain reaction, and co-immunoprecipitation assays. Via rescue experiments, it was concluded that AKR1B10 served as tumor suppressor potentially by ITGA5 expression in gastric cancer. Conclusion: Our results indicated that AKR1B10 inhibited migration, invasion, and adhesion of gastric cancer cells via modulation of ITGA5.


INTRODUCTION
Gastric cancer (GC) is a major cause of cancer-related deaths. 1 Metastasis of GC at early stage likely results in poor prognosis and high mortality. 2The research revealed that 5-year survival rate of advanced GC patients is less than 15%. 3 The complexity and multifactorial nature of metastasis mechanism in GC are considered as the main obstacles to promoting the survival of patients. 4Moreover, the molecular mechanism of GC development has not been clearly explored at present.Hence, exploring new molecular mechanisms of GC progression and finding new potential therapeutic targets are needed for improving the survival rate of GC patients.
Aldo-keto reductase 1B10 (AKR1B10) belongs to AKR superfamily.Aldo-keto reductase family are mainly soluble monomers with a molecular weight ranging from 34 to 37 kDa, and the AKR superfamily consists of nearly 200 members that can be classified into 16 categories with different subtypes in each category. 5A study verified that AKR members may participate in biological processes including carbonyl detoxification, hormone and lipid metabolism, osmoregulation, tumor formation, and tumor drug resistance in the organism. 6Aldo-keto reductase 1B10 participates in the occurrence of various tumors.Aldo-keto reductase 1B10 level is high in nongastrointestinal solid tumors like liver cancer, 7 lung cancer, 8 breast cancer, 9 and oral squamous cell carcinoma, 10 while it is decreased in colorectal cancer, 11 which may be caused by the tissue specificity of AKR1B10.Aldo-keto reductase 1B10 is mainly found in distal gastrointestinal tract, such as the small intestine and colorectum of healthy people. 12Aldo-keto reductase 1B10 can effectively catalyze the reduction of cytotoxic unsaturated carbonyl groups, thereby protecting the gastrointestinal tract from carboxyl damage and reducing the occurrence of tumors. 13Hence, we can infer that AKR1B10 acts as a tumor inhibitor in the occurrence and progression of gastrointestinal tumors, which is congruous with the previous finding that AKR1B10 is low in GC. 14 Nevertheless, the regulatory gene downstream of AKR1B10 and its mechanism in GC cells need further exploration.
Integrin subunit alpha 5 (ITGA5), a member of integrin family, is cancer-promoting by inducing communication between different cells or between cells and extracellular matrix. 15ITGA5 has been shown to produce an integral membrane protein to promote breast cancer cell metastasis. 16ITGA5 stimulates tumor progression and serves as an independent prognostic factor in esophageal squamous cell carcinoma. 17In addition, fibroblasts repressing colorectal adenocarcinoma require ITGA5, which is possible to be a stromal prognostic marker. 18But regulatory relationship of ITGA5 with AKR1B10 has not been investigated.
The main purpose of this work was to delineate expression levels of AKR1B10/ITGA5 and the specific mechanism of AKR1B10/ITGA5 in GC cell proliferation, migration, invasion, and cell adhesion through in vitro molecular and cellular assays, thereby assisting in finding new molecular targets for GC diagnosis and treatment.

MATERIALS AND METHODS Bioinformatics Method
Clinical sample profiles of The Cancer Genome Atlas-Stomach adenocarcinoma (TCGA-STAD) including 32 normal samples and 375 GC tissue samples were accessed from TCGA (https ://po rtal.gdc.c ancer .gov/ ) and were subjected to t-test for expression analysis of AKR1B10.The data were filtered out and processed via differential analysis by R package "edgeR" (|log fold change (FC)|>2, false discovery rate (FDR) < 0.05).Correlation analysis of AKR1B10 expression with clinical characteristics, and K-M analysis of AKR1B10 expression level and overall survival in GC were based on the TCGA database.Survival analyses were carried out using the "survival" package.

Clinical Samples
Gastric cancer (n = 20) and adjacent normal (n = 20) tissue samples of patients who did not undergo radiotherapy or chemotherapy before surgery were retrieved from Zhejiang Provincial People's Hospital, with all samples stored in liquid nitrogen.The study acquired approval from the Ethics Committee of Zhejiang Provincial People's Hospital (No: 2018KT050) and informed consent was obtained from each subject.
beads (Thermo Fisher Scientific) at 4°C.After beads were rinsed 4 times with pre-ice-cold lysis buffer, Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) sample buffer was supplemented for denaturation.AKR1B10 and ITGA5 protein levels were assayed via western blot.

RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction
Total RNA was isolated from tissues and cell lines with Trizol reagent (Invitrogen, USA, MA, Waltham).cDNA was synthesized by PrimeScript reverse transcriptase reagent kit (TaKaRa, Japan, Kyoto).Real-time quantitative polymerase chain reaction was done on synergy brandssynergy brands (SYBR) green PCR mix (TaKaRa) with glyce ralde hyde-3-pho sphat e dehydrogenase (GAPDH) as the internal reference.Primer sequences were listed in Table 1.Data were analyzed by applying 2 −ΔΔCt method.

Western Blot
After 24 hours of transfection, cells were rinsed with phosphate buffer solution (PBS) and lysed in the lysis buffer (Cell Signaling Technology, USA, MA, Boston) for 30 minutes.Protein concentration was identified with bicinchoninic acid protein assay kit (Pierce Biotechnology, USA, IL, Rockford).Proteins were subjected to SDS-PAGE and transferred onto a membrane.The membrane was sealed with 5% skim milk and then blotted overnight with primary antibodies anti-AKR1B10 (ab192865, 1:5000, Abcam), anti-ITGA5 (ab150361, 1:5000, Abcam), and anti-GAPDH (ab181602, 1:10 000, Abcam).After rinsing, the membrane was incubated with horseradish peroxidase-coupled secondary antibody IgG H&L (ab6721, 1:2500, Abcam) at room temperature for 1 hour.Signals were developed using enhanced chemiluminescence (ECL) detection reagent (Pierce) and the gray value was assessed on Image J software (Bethesda, Md, USA).GAPDH was utilized as a protein load control.
For detection of cell invasion, cell suspension of the same density was added in the upper chamber pre-coated with Matrigel and 600 μL medium plus 10% FBS in the lower chamber (Becton-Dickinson Biosciences) for 48 hours of incubation.After incubation, cells in the upper chamber were wiped off with a cotton swab, while cells on the other side were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet in 20% ethanol.The migrating and invading cells were monitored by photographs of 5 independent fields of each well using a LEICA microscope (Germany).This determination was conducted 3 times.

Wound Healing Assay
After transfection, cells were seeded on 6-well plates (2×10 5 cells/well) and grew to confluence.Monolayer was scratched with a 200 μL pipette tip and the floating cells were washed off with fresh medium, the remaining cells were followed by 24 hours of cell culture in an incubator under routine conditions.The wound areas were photographed at 0 and 24 hours for calculating the cell migration rate.The wound healing rate was computed as wound healing rate (%) = (wound width at 0 hour − wound width at 24 hours)/wound width at 0 hour × 100%.

Cell Adhesion Assay
To prevent non-specific cell adhesion, the 96-well plate was pre-treated with 10 μg/mL fibronectin (100 μL/well) (Becton-Dickinson Biosciences) at 37°C for 1 hour and treated with 1% bovine serum albumin (BSA) for 45 minutes.Firstly, cells were resuspended in a medium plus 1% FBS to adjust cell concentration.Cells were plated in the 96-well plate (2×10 4 cells/well) for 1 hour of incubation in the optimal incubator.Then, supernatant was discarded.Finally, cells were fixed for 15 minutes with 4% paraformaldehyde, stained with 0.2% crystal violet, counted, and pictured under a microscope.

Statistical Analysis
The relationship between AKR1B10 and ITGA5 in TCGA database and clinical samples was analyzed by the Pearson correlation.Measurement data were presented as the mean ± SD, and each experiment was completed at least 3 times.All data were subject to Student's t-test or 1-way analysis of variance on GraphPad Prism 5.0 (GraphPad Software, Inc., USA, CA, San Diego) and were then subject to Tukey's posthoc test for multiple comparisons.P < .05means a statistically significant difference.

Aldo Keto Reductase 1B10 Expression Is Noticeably Lower in Gastric Cancer
In the previous reviews, our researchers found that AKR1B10 is a favorable prognostic indicator in GC and remarkably downregulated in clinical samples. 14herefore, this study mainly probed the specific relevant molecular mechanism of AKR1B10 in the occurrence and metastasis of GC.Firstly, bioinformatics methods were used to analyze data in TCGA-STAD dataset.The AKR1B10 level in GC tissues was dramatically lower than that in normal tissues (Figure 1A).Also, correlations between AKR1B10 expression levels and clinical characteristics as well as overall survival were analyzed.The results showed no significant differences in AKR1B10 expression among T classification (P = .19),N classification (P = .93),M classification (P = .44),clinical stage (P = .69),and overall survival (Supplementary Figure 1A-E).
Real-time quantitative polymerase chain reaction was implemented to quantify AKR1B10 level in 20 pairs of clinical samples of GC tissue and adjacent normal tissue, which manifested that AKR1B10 level in the clinical samples of GC tissue showed the same trend as predicted by bioinformatics analysis (Figure 1B).Subsequently, mRNA and protein levels of AKR1B10 in GES-1, SGC-7901, BGC-823, MKN-45, and AGS cell lines were assayed via qRT-PCR and western blot.The AKR1B10 mRNA and protein levels were markedly reduced in GC cell lines (Figure 1C and 1D).Overall, AKR1B10 in GC tissues and cells was relatively less expressed.Then, SGC-7901 cell line with the lowest level of AKR1B10 and AGS cell line with the highest level of AKR1B10 were chosen for subsequent experiments.

Aldo-Keto Reductase 1B10 Restrains Cell Viability, Migration, Invasion, and Adhesion of Gastric Cancer Cells In Vitro
To measure the impact of AKR1B10 on cell viability, migration, invasion, and adhesion of GC cells in vitro, oe-AKR1B10 was transfected into SGC-7901 cells and si-AKR1B10 was transfected into AGS cells, respectively.Transfection efficiencies were assayed through qRT-PCR, and results indicated that AKR1B10 level was noticeably up-regulated after transfecting with oe-AKR1B10 and downregulated after transfecting with si-AKR1B10 (Figure 2A).Cell Counting Kit-8 assay verified that overexpressing AKR1B10 remarkably inhibited cell viability, but the si-AKR1B10 group exerted the opposite effect (Figure 2B).Transwell migration and invasion assays substantiated that overexpressing AKR1B10 conspicuously hindered cell migratory and invasive abilities, and silencing AKR1B10 promoted the above phenotypes (Figure 2C).As such, the wound healing assay also ascertained that overexpressing AKR1B10 could noticeably hamper cell migratory ability (Figure 2D).Cell adhesion assay displayed that overexpressing AKR1B10 notably reduced cell adhesive ability (Figure 2E).However, knockdown of AKR1B10 fostered cell migration and adhesion of GC cells.Collectively, AKR1B10 served as tumor suppressor in GC.

Overexpression of Aldo-keto Reductase 1B10 Inhibits the Expression of Integrin Subunit Alpha 5
Based on the previous research, 19 we conjectured that ITGA5 may be a downstream regulatory gene of AKR1B10.
The expression analysis results of ITGA5 in clinical samples detected by qRT-PCR were shown in Figure 3A, which denoted that ITGA5 expression in tumor tissues (n = 20) was prominently higher than that in adjacent normal tissues (n = 20).mRNA and protein levels of ITGA5 in GES-1, SGC-7901, BGC-823, MKN-45, and AGS cell lines were assayed through qRT-PCR and western blot, respectively.In accordance with results in clinical samples, mRNA and protein levels of ITGA5 in GC cells were dramatically higher than in normal gastric mucosal cells (Figure 3B).In the analysis of clinical samples and TCGA database, ITGA5 was negatively correlated with AKR1B10 (Figure 3C and 3D).Likewise, ITGA5 mRNA level was noticeably reduced in oe-AKR1B10 group (Figure 3E), which suggested that AKR1B10 suppressed the expression of ITGA5.Interaction of AKR1B10 with ITGA5 was confirmed using Co-IP assays (Figure 3F), which confirmed the binding relationship of AKR1B10 with ITGA5.Thus, ITGA5 was upregulated in GC and negatively regulated by AKR1B10.

Aldo-keto Reductase 1B10 Regulates Integrin Subunit Alpha 5 to Function in Gastric Cells
To investigate the impact of AKR1B10 on GC cells (SGC-7901) by regulating ITGA5, a rescue experiment was carried out and 3 groups were constructed: vector [(ove rexpr essin g)oe-NC(AKR1B10)+oe-NC(ITGA5)], oe-AKR1B10 [oe-AKR1B10 + oe-NC(ITGA5)], and oe-AKR1B10 + oe-ITGA5.Firstly, transfection efficiency was validated by qRT-PCR and western blot.Then, qRT-PCR and western blot respectively manifested that ITGA5 mRNA and protein levels decreased substantially in oe-AKR1B10 group, whereas their expression levels were restored in oe-AKR1B10+oe-ITGA5 group (Figure 4A).Through CCK-8 assay, it was found that overexpressing ITGA5 and AKR1B10 simultaneously could neutralize the repressive effect of overexpressing AKR1B10 on cell viability (Figure 4B).Migratory and invasive potentials of SGC-7901 cells were attenuated in oe-AKR1B10 group, but this effect was offset by overexpressing ITGA5 and AKR1B10 simultaneously (Figure 4C and 4D).Cell adhesion assay also revealed a consistent trend that overexpressing AKR1B10 and ITGA5 simultaneously abrogated the inhibition of cell adhesion by overexpressing AKR1B10 (Figure 4E).Therefore, AKR1B10 could suppress proliferation, migration, invasion, and adhesion of GC cells by modulating ITGA5.

DISCUSSION
Since GC is a common malignancy, it is of great significance for improvement of treatment and patient's prognosis to clarify its occurrence and progression mechanism.AKR1B10 is known to be a good clinical and prognostic predictor for GC 14,20 and affects doxorubicin resistance and cisplatin resistance of GC cells. 21,22Shen et al 23 established a mice model and found increased gene mutations and dysplasia of intestinal mucosal cells in mice deficient in AKR1B8 (the ortholog of human AKR1B10).
Numerous studies substantiated that AKR1B10 expression is low in GC. [24][25][26] Shao et al 17 reported that AKR1B10 was lowly expressed in GC tissues and repressed proliferation, migration, and invasion of GC cells.This study also manifested that AKR1B10 levels in GC tissues and cells were conspicuously lower in comparison to normal tissues and cells through analyzing TCGA-STAD dataset and detecting the AKR1B10 expression in GC tissues and cell lines in vitro, which was in line with the earlier studies.AKR1B10 plays a pro-cancer role in varying cancer species.Qu et al 18 reported that AKR1B10 was substantially upregulated in breast cancer tissues, and it facilitated cell malignant phenotypes via modulation of Phosphoinositide 3-kinase/Protein Kinase B/Nuclear factor kappa-B (PI3K/AKT/NF-κB) signaling pathway.Huang et al 27 revealed that AKR1B10 was highly expressed in bladder cancer tissues and modulated the proliferation, migration, invasion, and cell stemness of bladder cancer  An existing study showed that AKR1B10 can regulate the expression of ITGA5 in breast cancer. 19ITGA5 mainly distributes in fibroblasts and is connected to fibronectin.ITGA5 is involved in varying cancers.For instance, in esophageal squamous cell carcinoma, ITGA5 can promote cell migration and cisplatin resistance. 29ITGA5 can increase the survival rate and metastatic potential of lung cancer cells. 30ITGA5 promotes GC cell proliferation, migration, invasion, and tumor growth in vivo by focal adhesion kinase (FAK)-AKT signaling pathway. 31ITGA5 fosters GC cell proliferation, migration, invasion, and metastasis in vivo by inducing the epith elial -to-m esenc hymal transition process. 32This study carried out a bioinformatics analysis to reveal that ITGA5 was negatively correlated with AKR1B10, and ITGA5 was increased in GC tissues and cells.The result was in line with a result in an earlier study. 31Afterward, in vitro rescue experiments in cells justified that AKR1B10 suppressed GC cell viability, migration, invasion, and adhesion by regulating ITGA5, which further verified the regulatory relationship between AKR1B10 and ITGA5.
However, these results had certain limitations.For instance, the regulatory mechanism between AKR1B10 and ITGA5 was not certified in vivo nor was the specific downstream regulatory mechanism of ITGA5 in regulating cell biological functions.These deficiencies would be developed in further research.

CONCLUSION
All in all, this study verified that AKR1B10 was notably downregulated, while ITGA5 was markedly upregulated in GC, and AKR1B10 hampered GC cell viability, invasion, migration, and adhesion by regulating ITGA5, through analysis of TCGA data along with clinical data, as well as in vitro molecular and cell experiments.These findings lay a theoretical foundation for exploring novel molecules for clinical diagnosis and treatment of GC and generate fresh insight into finding safer and more effective treatments for GC.

Figure 2 .
Figure 2. AKR1B10 restrains cell viability, invasion, migration, and adhesion of GC in vitro.(A) AKR1B10 mRNA expression in each transfection group was assayed via qRT-PCR; (B) GC cell viability in different treatment groups was measured via CCK-8 assay; (C) migratory and invasive abilities of GC cells in different treatment groups were assessed through transwell assays (×100); (D) migratory ability of GC cells in different treatment groups were assayed through wound healing assay (×40); (E) the adhesive ability of GC cells in different treatment groups was detected by cell adhesion assay (×100); * P < .05.AKR1B10, aldo-keto reductase 1B10; CCK-8, Cell Counting Kit-8; GC, gastric cancer; qRT-PCR, real-time quantitative polymerase chain reaction.

Figure 4 .
Figure 4. Aldo-keto reductase 1B10 regulates ITGA5 to function in GC cells.(A) Integrin subunit alpha 5 mRNA and protein levels in different treatment groups were detected via qRT-PCR and western blot, respectively; (B) Cell viability in different treatment groups was detected via CCK-8 assay; (C) Migratory and invasion abilities in different treatment groups were tested through Transwell assay (×100); (D) Migratory ability in different treatment groups was detected via wound healing assay (×40); (E) Cell adhesive property in different treatment groups was tested by cell adhesion assay (×100); * P < .05.AKR1B10, aldo-keto reductase 1B10; CCK-8, Cell Counting Kit-8; ITGA5, integrin subunit alpha 5; qRT-PCR, real-time quantitative polymerase chain reaction; oe, overexpressing.